Substituted indole derivatives

ABSTRACT

The present invention relates to substituted indole derivatives, to processes for their production, their use as pharmaceuticals and to pharmaceutical compositions comprising them.

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/486,808 filed May 17, 2011; the contentsof which is incorporated herein by reference in their entirety.

The present invention relates to substituted indole derivatives, toprocesses for their production, their use as pharmaceuticals and topharmaceutical compositions comprising them.

BACKGROUND OF THE INVENTION

Poor bioavailability of drug products is frequently a limiting factorfor pharmaceutically effective ingredients. This problem is nowaddressed in a particular field of indole derivatives by converting acorresponding parent drug into a derivative thereof, which appears tohave unexpected favorable effects as compared to its parent compounds.

SUMMARY OF THE INVENTION

Specifically, the present invention relates to a compound of formula (I)or a pharmaceutically acceptable salt or hydrate thereof,

whereinX is CH or N;R is H or PO₃H₂;R1 is H; or C₁₋₄alkyl;R2 is H; or C₁₋₄alkyl;R3 is H; C₁₋₄alkyl; CN; Hal; or OH; andR4 and R5 are independently from each other H, or C₁₋₄alkyl; or R4 andR5 form together with the carbon atom to which they are attached a 3-6membered cycloalkyl group.

In another embodiment the present invention relates to a compound offormula (I) or a pharmaceutically acceptable salt or hydrate thereof,

wherein X is CH;

R is PO₃H₂;

R1 is H;

R2 is H; or C₁₋₄alkyl;

R3 is H; or C₁₋₄alkyl; and

R4 and R5 are independently from each other H; or R4 and R5 formtogether with the carbon atom to which they are attached a 3-6 memberedcycloalkyl group.

In another embodiment the present invention relates to a compound offormula (I) or a pharmaceutically acceptable salt or hydrate thereof,

wherein X is CH;

R is H;

R1 is H;

R2 is H; or C₁₋₄alkyl;

R3 is H; or C₁₋₄alkyl; and

R4 and R5 are independently from each other H; or R4 and R5 formtogether with the carbon atom to which they are attached a 3-6 memberedcycloalkyl group.

In another embodiment the present invention relates to a compound offormula (I) or a pharmaceutically acceptable salt or hydrate thereof,

wherein X is N;

R is PO₃H₂;

R1 is H;

R2 is H; or C₁₋₄alkyl;

R3 is H; and

R4 and R5 are independently from each other H; or R4 and R5 formtogether with the carbon atom to which they are attached a 3-6 memberedcycloalkyl group.

In another embodiment the present invention relates to a compound offormula (I) or a pharmaceutically acceptable salt or hydrate thereof,

wherein X is N;

R is PO₃H₂;

R1 is H;

R2 is H; or C₁₋₄alkyl;

R3 is H; and

R4 and R5 are independently from each other H; or C₁₋₄alkyl.

In another embodiment the invention relates to a compound of formula(II)

or a pharmaceutically acceptable salt thereof.

In another embodiment the invention relates to a compound of formula(III)

or a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment the invention relates to a compound of formula(IV)

or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the X-ray diffractogram of the crystalline monohydrate ofphosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester(monohydrate of Example No 1).

FIG. 2 shows the water absorption profile of crystalline Example No. 1mono-hydrate [6 h equilibration at 50% RH, followed by 2 RH cycles from50% RH to 90% RH to 0% RH to 90% RH to 0% RH to 50% RH in 10% RH steps].RH=Relative Humidity.

PRIOR ART

N. Fotouhi et al. (EP 1,224,181) describe substituted pyrrolederivatives wherein the chemical modification on said pyrrole ringconsists of a large number of variables and may also contain a methylenehydroxy or a methylene phosphate group.

DEFINITIONS

As used herein, the term “halogen” (or halo) refers to fluorine,bromine, chlorine or iodine, in particular fluorine, chlorine.

As used herein, the term “alkyl” refers to a fully saturated branched orunbranched hydrocarbon moiety having up to 4 carbon atoms.Representative examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl and the like.

As used herein, the term “alkoxy” refers to alkyl-O—, wherein alkyl isdefined herein above. Representative examples of alkoxy include, but arenot limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy,term-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy- andthe like. Typically, alkoxy groups have 1-4 carbon atoms.

As used herein, the term “cycloalkyl” refers to saturated or unsaturatedmonocyclic hydrocarbon groups of 3-6 carbon atoms, particularly 3-5carbon atoms, especially 3-4 or 3 carbon atoms.

As used herein, the terms “salt” or “salts” refers to an acid additionor base addition salt of a compound of the invention. “Salts” include inparticular “pharmaceutical acceptable salts”. The term “pharmaceuticallyacceptable salts” refers to salts that retain the biologicaleffectiveness and properties of the compounds of this invention and,which typically are not biologically or otherwise undesirable. In manycases, the compounds of the present invention are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids, e.g., acetate, aspartate, benzoate,besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulformate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example,acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,toluenesulfonic acid, sulfosalicylic acid, and the like.Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, cholinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound, a basic or acidic moiety, byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, use of non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile isdesirable, where practicable. Lists of additional suitable salts can befound, e.g., in “Remington's Pharmaceutical Sciences”, 20th ed., MackPublishing Company, Easton, Pa., (1985); and in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F ³¹P, ³²P, ³⁵S, ³⁶Cl, ¹²⁵Irespectively. The invention includes various isotopically labeledcompounds as defined herein, for example those into which radioactiveisotopes, such as ³H, and ¹⁴C, or those into which non-radioactiveisotopes, such as ²H and ¹³C are present. Such isotopically labelledcompounds are useful in metabolic studies (with ¹⁴C), reaction kineticstudies (with, for example ²H or ³H), detection or imaging techniques,such as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT) including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Inparticular, an ¹⁸F or labeled compound may be particularly desirable forPET or SPECT studies. Isotopically labeled compounds of this inventionand prodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent.

Further, substitution with heavier isotopes, particularly deuterium(i.e., ²H or D) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements or an improvement in therapeutic index. Itis understood that deuterium in this context is regarded as asubstituent of a compound of the invention. The concentration of such aheavier isotope, specifically deuterium, may be defined by the isotopicenrichment factor. The term “isotopic enrichment factor” as used hereinmeans the ratio between the isotopic abundance and the natural abundanceof a specified isotope. If a substituent in a compound of this inventionis denoted deuterium, such compound has an isotopic enrichment factorfor each designated deuterium atom of at least 3500 (52.5% deuteriumincorporation at each designated deuterium atom), at least 4000 (60%deuterium incorporation), at least 4500 (67.5% deuterium incorporation),at least 5000 (75% deuterium incorporation), at least 5500 (82.5%deuterium incorporation), at least 6000 (90% deuterium incorporation),at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97%deuterium incorporation), at least 6600 (99% deuterium incorporation),or at least 6633.3 (99.5% deuterium incorporation).

Isotopically-labeled compounds of the invention, can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described in the accompanying Examplesand Preparations using an appropriate isotopically-labeled reagents inplace of the non-labeled reagent previously employed.

Pharmaceutically acceptable solvates in accordance with the inventioninclude those wherein the solvent of crystallization may be isotopicallysubstituted, e.g. D₂O, d₆-acetone, d₆-DMSO.

Compounds of the invention, e.g. compounds of formula (I) that containgroups capable of acting as donors and/or acceptors for hydrogen bondsmay be capable of forming co-crystals with suitable co-crystal formers.These co-crystals may be prepared from compounds of formula (I) by knownco-crystal forming procedures. Such procedures include grinding,heating, co-subliming, co-melting, or contacting in solution compoundsof formula (I) with the co-crystal former under crystallizationconditions and isolating co-crystals thereby formed. Suitable co-crystalformers include those described in WO 2004/078163. Hence the inventionfurther provides co-crystals comprising a compound of formula (I).

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated.

The term “a therapeutically effective amount” of a compound of thepresent invention refers to an amount of the compound of the presentinvention that will elicit the biological or medical response of asubject, for example, reduction or inhibition of an enzyme or a proteinactivity, or ameliorate symptoms, alleviate conditions, slow or delaydisease progression, or prevent a disease, etc. In one non-limitingembodiment, the term “a therapeutically effective amount” refers to theamount of the compound of the present invention that, when administeredto a subject, is effective to (1) at least partially alleviating,inhibiting, preventing and/or ameliorating a condition, or a disorder ora disease (i) mediated by protein kinase C, or (ii) associated withprotein kinase C activity, or (iii) characterized by activity (normal orabnormal) of protein kinase C; or (2) reducing or inhibiting theactivity of protein kinase C; or (3) reducing or inhibiting theexpression of protein kinase C. In another non-limiting embodiment, theterm “a therapeutically effective amount” refers to the amount of thecompound of the present invention that, when administered to a cell, ora tissue, or a non-cellular biological material, or a medium, iseffective to at least partially reducing or inhibiting the activity ofprotein kinase C; or at least partially reducing or inhibiting theexpression of protein kinase C.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans, male or female), cows, sheep, goats, horses, dogs, cats,rabbits, rats, mice, fish, birds and the like. In certain embodiments,the subject is a primate. In yet other embodiments, the subject is ahuman.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat”, “treating” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

Methods of Manufacturing

The compounds of the invention may be manufactured by the methodsprovided below, e.g. by converting maleimide of formula (Va) into thealcohol of formula (Vb) for example with formaldehyde in the presence orabsence of a solvent or a base such as potassium carbonate andoptionally prior to this reaction by introducing protective groups, e.g.tert-butoxycarbonyl groups in accordance to the state-of-the-artreaction, for example when there are free and reactive amino-groups in acompound of formula (Va), wherein the variables, X, R, R1, R2, R3, R4and R5 have the meanings as provided for formula (I).

The alcohol of formula (Vb) may be optionally converted to a reactiveester, e.g. with trichloromethylacetonitril and appropriate base, e.g.DBU, and may then be reacted with an appropriated phosphorylating agent,e.g. phosphoric acid di-tert-butylester in the presence or absence of anappropriate solvent, e.g. an aprotic solvent, e.g. acetonitril, and maythen be hydrolysed, e.g. with trifluoroacetic acid e.g. indichloromethane or 1,2-dichloroethane to furnish the final product Vc.

Alternatively, the alcohol of formula (Vb) may be reacted directly witha phosphoric acid ester, e.g. with phosphoric acid di-tert-butylester,e.g. under Mitsunobu reaction conditions to furnish the phosphoric acidester, which may then be hydrolised, e.g. with trifluoroacetic acid,e.g. in dichloromethane to furnish the final product Vc.

Experimental Part

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or may be prepared analogously tomethods known in the art or as described hereafter.

The following examples are illustrative of the invention without anylimitation.

ABBREVIATIONS

bs broad singlet

d doublet

DMSO dimethylsulfoxide

d.n. dose normalized

EtOAc ethyl acetate

Et₂O diethyl ether

FCC flash column chromatography

MeOH methanol

MS mass spectroscopy

m multiplet

NMR nuclear magnetic resonance

p.o. per os

r.t. room temperature

s singlet

t triplet

TFA trifluoroacetic acid

TLC thin layer chromatography

UPLC ultra high pressure liquid chromatography

The chemical nomenclature of all compounds was created by usingAutoNom®.

NMR spectra were recorded on a Bruker Avance DPX 400 spectrometer atroom temperature.

LCMS Methods Used:

LC Method 1 (Rt⁽¹⁾):

The retention times (Rt) were obtained on a Waters Acquity UPLC systemlinked to a Waters ZQ 2000 mass spectrometer using a Waters BEH C18 1.7μm 2.1×50 mm column (flow rate=0.7 ml/min; detection 240-350 nm; DAD)applying a gradient (solvent A: water+0.1% formic acid, solvent B:acetonitrile; t=0 min: 99% A, 1% B; t=1 min 98% A, 2% B; t=2.25 min 1%A, 99% B; t=4.5 min 0% A, 100% B).

LC Method 2 (Rt⁽²⁾):

The retention times (Rt) were obtained on a Agilent HPLC system with anAscentis® Express column C18 2.7 μm, 30×2.1 mm (Supelco) applying agradient (H₂O+0.05% formic acid+3.75 mM Ammonium acetate)/(CH₃CN+0.04%formic acid) 90/10 to 5/95 over 1.7 min and 1.2 mL/min as solvent flowand then 5/95 over 0.7 min with 1.4 mL/min as solvent flow and 40° C.for the oven temperature. Detection method UV 214-350 nm-MS.

Purification Method:

Preparative Reverse Phase Gilson HPLC

Column SunFire prep C18 OBD 5 μm, 30×100 mm from WATERS, with H₂O+0.1%TFA and Acetonitrile+0.1% TFA as mobile phase. Detection method UV220-400 nm

EXAMPLE 1 Phosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester

To a solution of7-{1-[1-(di-tert-butoxy-phosphoryloxymethyl)-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-isoquinolin-3-yl}-4,7-diaza-spiro[2.5]octane-4-carboxylicacid tert-butyl ester (1.90 g, 2.42 mmol) in 1,2-dichloroethane (50 mL)was added TFA (8.27 g, 72.5 mmol) under argon at 0° C. The reactionmixture was stirred for 3.5 h under argon at 0° C. until UPLC-MSindicated that full conversion of the starting material had occurred.The reaction mixture was diluted with 1,2-dichloroethane (50 mL) andconcentrated at reduced pressure to afford the crude product as a redsolid. The crude reaction product was dissolved in MeOH and slowlyconcentrated at reduced pressure until crystallization started to occur.Pentane was added and the solids were filtered off and washed with Et₂O.Further purification was achieved by suspending the crude product inDMSO, followed by sonication for 30 min. The solids were filtered off,washed with Et₂O and dried at high vacuum (<1 mm Hg) to afford the titlecompound as a dark red solid. ¹H-NMR (400 MHz, DMSO-d6): 12.10 (s, 1H),8.08 (d, 1H), 7.65-7.61 (m, 2H), 7.44 (t, 1H), 7.20 (s, 1H), 7.06 (t,1H), 6.74 (d, 1H), 6.43 (t, 1H), 6.00 (d, 1H), 5.29 (d, 2H), 3.87-3.01(m, 6H), 2.37 (s, 3H), 0.97-0.62 (m, 4H). ³¹P-NMR (162 MHz, DMSO-d6):−6.0. LCMS: [M+1]⁺=574.0, Rt⁽¹⁾=1.77 min., Rt⁽²⁾=0.71 min.

Preparation of7-{1-[4-(7-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-isoquinolin-3-yl}-4,7-diaza-spiro[2.5]octane-4-carboxylicacid tert-butyl ester

To a solution of7-{1-[1-hydroxymethyl-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-isoquinolin-3-yl}-4,7-diaza-spiro[2.5]octane-4-carboxylicacid tert-butyl ester (7.30 g, 12.30 mmol) in acetonitrile (60 mL) wasadded dropwise a solution of DBU (0.374 g, 0.371 mL, 2.46 mmol) intrichloroacetonitrile (17.8 g, 12.3 mL, 123 mmol) at r.t. under argon.The reaction mixture was stirred for 5 h at r.t. until TLC (SiO₂,EtOAc/cyclohexane 6:4) indicated complete conversion. The reactionmixture was evaporated to dryness at reduced pressure and the residuewas suspended in acetonitrile (60 mL). Phosphoric acid di-tert-butylester (3.36 g, 15.99 mmol) was added and the reaction mixture wasstirred for approximately 3.5 h at r.t. under argon until TLC (SiO₂,EtOAc/cyclohexane 6:4) indicated that the reaction was complete. Thereaction mixture was concentrated at reduced pressure and the residuewas partitioned between EtOAc and water. The layers were separated andthe organic phase was washed with water (5 times). The organic phase wasdried over Na₂SO₄ and concentrated at reduced pressure to afford a redsolid. The crude product was purified by FCC (Biotage SP4™ system, SiO₂,cyclohexane/EtOAc 20:80) to yield the title compound as a red solid.¹H-NMR (400 MHz, DMSO-d6): 12.03 (s, 1H), 8.08 (d, 1H), 7.70-7.65 (m,2H), 7.47 (t, 1H), 7.11 (s, 1H), 7.11-7.07 (m, 1H), 6.77 (d, 1H), 6.44(t, 1H), 5.97 (d, 1H), 5.39 (d, 1H), 3.49-3.08 (m, 6H), 2.39 (s, 3H),1.45 (s, 18H), 1.41 (s, 9H), 0.87-0.56 (m, 4H). ³¹P-NMR (162 MHz,DMSO-d6): −12.1. LCMS: [M+1]⁺=786.4, Rt⁽¹⁾=2.53 min., Rt⁽²⁾=1.62 min.

Preparation of7-{1-[1-Hydroxymethyl-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-isoquinolin-3-yl}-4,7-diaza-spiro[2.5]octane-4-carboxylicacid tert-butyl ester

To a solution of7-{1-[4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-isoquinolin-3-yl}-4,7-diaza-spiro[2.5]octane-4-carboxylicacid tert-butyl ester (3.00 g, 5.32 mmol) in MeOH (25 mL) was added anaqueous 37% solution of formaldehyde (9.5 g, 8.72 mL, 117 mmol) underargon at r.t. The reaction mixture was heated to 85° C. and stirred for4 h. The reaction mixture was cooled to r.t. under continuous stirringand filtered. The solids were washed with ice-water and dried at highvacuum (<1 mm Hg) to afford the title compound as dark red crystals.¹H-NMR (400 MHz, DMSO-d6): 11.96 (s, 1H), 8.05 (s, 1H), 7.69-7.64 (m,2H), 7.47 (t, 1H), 7.12-7.06 (m, 2H), 6.76 (d, 1H), 6.42 (t, 1H), 6.01(d, 1H), 5.00 (d, 2H), 3.49-3.10 (m, 6H), 2.39 (s, 3H), 1.41 (s, 9H),0.89-0.57 (m, 4H). LCMS: [M]⁺=593.7, Rt⁽¹⁾=2.34 min.

Preparation of7-{1-[4-(7-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-isoquinolin-3-yl}-4,7-diaza-spiro[2.5]octane-4-carboxylicacid tert-butyl ester

Di-tert-butyl dicarbonate (2.77 g, 10.8 mmol) and triethyl amine (2.18g, 21.6 mmol) were added to a solution of3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-pyrrole-2,5-dione(5.0 g, 10.8 mmol) in THF (50 mL) under argon at r.t. The reactionmixture was stirred for 16 h and concentrated at reduced pressure. Theresidue was partitioned between a saturated aqueous NH₄Cl solution andCH₂Cl₂. The layers were partitioned and the aqueous layer was extractedwith CH₂Cl₂. The combined organic layers were washed with a saturatedaqueous NaHCO₃ solution and brine, dried over anhydrous Na₂SO₄ andconcentrated at reduced pressure to afford the title compound as anorange solid. ¹H-NMR (400 MHz, DMSO-d6): 11.88 (s, 1H), 11.15 (s, 1H),8.00 (d, 1H), 7.68-7.65 (m, 2H), 7.46 (t, 1H), 7.09 (t, 1H), 7.06 (s,1H), 6.73 (d, 1H), 6.41 (t, 1H), 6.01 (d, 1H), 3.48-3.16 (m, 6H), 2.38(s, 3H), 1.40 (s, 9H), 0.87-0.54 (m, 4H). LCMS: [M+1]⁺=563.9, Rt⁽¹⁾=3.51min, Rt⁽¹⁾=2.36 min., Rt⁽²⁾=1.37 min.

EXAMPLE 23-[3-(4,7-Diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-1-hydroxymethyl-4-(7-methyl-1H-indol-3-yl)-pyrrole-2,5-dione

To a solution of7-{1-[1-hydroxymethyl-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-isoquinolin-3-yl}-4,7-diaza-spiro[2.5]octane-4-carboxylicacid tert-butyl ester (500 mg, 0.842 mmol) in 1,2-dichloroethane (5 mL)was added TFA (1.44 g, 0.973 mL, 12.7 mmol) under argon at 0° C. Thereaction mixture was stirred for 1 h under argon at 0° C. after whichadditional TFA (0.768 g, 0.52 mL, 6.74 mmol) was added. Stirring wascontinued for 1.5 h at 0° C. The reaction mixture was evaporated todryness at reduced pressure and the crude product was crystallised fromMeOH to afford the title compound as a red solid (TFA salt) as a darkred solid. ¹H-NMR (400 MHz, DMSO-d6): 12.01 (d, 1H), 9.06 (bs, 2H), 8.11(d, 1H), 7.71-7.66 (m, 2H), 7.51 (t, 1H), 7.27 (s, 1H), 7.14 (t, 1H),6.77 (d, 1H), 6.45-6.39 (m, 2H), 5.92 (d, 1H), 5.00 (d, 2H), 3.84-3.54(m, 6H), 2.39 (s, 3H), 0.96-0.67 (m, 4H). LCMS: [M+1]⁺=493.7, Rt⁽¹⁾=1.84min.

EXAMPLE 33-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-pyrrole-2,5-dione

The synthesis of the title compound has been described as Example 69 inWO03082859.

EXAMPLE 4 Phosphoric acidmono-{3-(1H-indol-3-yl)-4-[2-(4-methyl-piperazin-1-yl)-quinazolin-4-yl]-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl}ester

Under argon, phosphoric acid di-tert-butyl ester chloromethyl ester(1.24 g, 4.81 mmol) and Cs₂CO₃ (3.14 g, 9.63 mmol) were added to asolution of3-(1H-indol-3-yl)-4-[2-(4-methyl-piperazin-1-yl)-quinazolin-4-yl]-pyrrole-2,5-dione(2.0 g, 4.01 mmol) in acetone (40 mL). The reaction mixture was stirredunder argon for 16 h at 50° C. followed by concentration at reducedpressure. The residue was partitioned between EtOAc and a saturatedaqueous NH₄Cl solution, the layers were separated and the organic layerwas dried over anhydrous Na₂SO₄. Concentration at reduced pressureafforded the crude product as a red foam. The crude product was purifiedby reverse phase Gilson HPLC as described above. After concentration ofthe desired fractions in vacuo, a red solid was obtained. UPLC-MSindicated that partial cleavage of the t-butyl ester groups hadoccurred. The thus obtained mixture (280 mg) was dissolved in a mixtureof 1,2-dichloroethane (4 mL) and acetonitrile (2.0 mL). TFA (145 mg, 98μL, 1.27 mmol) was added and the resulting solution was stirred for 3 hunder argon at 0° C. when UPLC-MS indicated that full conversion of thestarting material had occurred. The reaction mixture was diluted with1,2-dichloroethane (4.0 mL) and concentrated at reduced pressure toafford the crude product as a red solid. The crude reaction product wasdissolved in MeOH (3 mL) and slowly concentrated at reduced pressureuntil crystallization started to occur. The crystals were filtered offand washed with Et₂O and pentane to afford the title compound as anorange solid. ¹H-NMR (400 MHz, DMSO-d6): 12.27 (s, 1H), 8.21 (s, 1H),7.72-7.66 (m, 2H), 7.58 (d, 1H), 7.40 (d, 1H), 7.14 (t, 1H), 7.02 (t,1H), 6.65 (t, 1H), 6.24 (d, 1H), 5.34 (d, 2H), 4.14-3.72 (bs, 4H),3.00-2.73 (bs, 4H), 2.60 (s, 3H). ³¹P-NMR (162 MHz, DMSO-d6): −2.7.LCMS: [M]⁺=548.6, Rt⁽¹⁾=1.72 min., Rt⁽²⁾=0.73 min.

Preparation of Crystalline Material of the Compound of Example 1

2 grams of phosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester(example No. 1) were dispersed in a mixture of 800 ml ethanol and 200 mlof water. This suspension was stirred at room temperature for 3 days.Thereafter, said suspension was filtered through a sintered glass filterand the crystals so obtained were dried in normal atmospheric airstream. To 1 gram of these crystals, 4 ml of a solution of 80%ethanol/20% water (vol./vol.) was added and the resulting mixture wasevaporated to dryness at atmospheric pressure to furnish themono-hydrate of example No. 1.

FIG. 1

An X-ray diffractogram of the above crystalline monohydrate is shown inFIG. 1, and peaks are recorded against the angle 2 theta in table 1.

TABLE 1 Main peaks on the X-Ray diffraction pattern of the crystallinemono-hydrate of example No. 1 d value Intensity Angle 2θ Angstrom CountsCount % Intensity Intensity % 8.506 10.38645 8.506 10.38645 5215 26.09.525 9.27790 9.525 9.27790 7052 35.2 13.793 6.41501 13.793 6.41501 424521.2 14.926 5.93044 14.926 5.93044 3513 17.5 15.172 5.83502 15.1725.83502 3360 16.8 15.413 5.74440 15.413 5.74440 3384 16.9 16.356 5.4151916.356 5.41519 6738 33.6 17.091 5.18400 17.091 5.18400 20046 100 18.0054.92268 18.005 4.92268 10587 52.8 19.224 4.61338 19.224 4.61338 644232.1 20.859 4.25526 20.859 4.25526 9742 48.6 22.433 3.96009 22.4333.96009 5332 26.6 23.316 3.81209 23.316 3.81209 5751 28.7 25.792 3.4514025.792 3.45140 8574 42.8 27.402 3.25222 27.402 3.25222 3930 19.6 27.7123.21657 27.712 3.21657 3575 17.8 28.091 3.17398 28.091 3.17398 4187 20.930.521 2.92662 30.521 2.92662 2899 14.5 31.502 2.83768 31.502 2.837683756 18.7

Accordingly, the present invention provides in another embodiment acrystalline form of phosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester,especially the mono-hydrate, which preferably has an X-ray powderdiffraction pattern with at least one, preferably two, more preferablythree, even more preferably four, especially five, most preferably allof the following peaks at an angle of refraction 2 theta (θ) of 9.525,16.356, 17.091, 18.005, 20.859, each ±0.2, especially as depicted inFIG. 1.

TABLE 2 Chemical stability of amorphous and crystalline example No. 1Initial Purity Temperature Exposure Purity Compound Solid State (%) [°C.] Time [%] Example Amorphous 98.5 50 1 week 95.8 No. 1 ExampleAmorphous 98.5 80 1 week 89.7 No. 1 Example Crystalline 100 50 1 week100 No. 1 mono- hydrate Example Crystalline 100 80 1 week 100 No. 1mono- hydrate

Table summarizing the thermal events seen on DSC (differential scanningcalorimetry) and TGA (thermal gravimetric analysis)

Temperature range [° C.] Event  30-125 Loss of water 225-265DegradrationFIG. 2

The water sorption profile of the crystalline Example No. 1 mono-hydrateis shown in FIG. 2. The following humidity exposure is applied: 6 hequilibration at 50% RH, followed by 2 RH cycles from 50% RH to 90% RHto 0% RH to 90% RH to 0% RH to 50% RH in 10% RH steps. (RH=relativehumidity)

Biopharmaceutical Part

The compounds of the invention, for example a compound of formulae (I),(II), (III) or (IV) and the like in free form or in pharmaceuticallyacceptable salt or hydrate form, exhibit valuable pharmacologicalproperties as described in the tests below, e.g. in vitro and in vivotests, and are therefore indicated for therapy.

A. In Vitro

1. Protein Kinase C Alpha and Theta Assays

The compounds of the invention were tested for their activity ondifferent PKC isotypes according to the following method. All assayswere performed in 384 well microtiter plates. Each assay plate contained8-point serial dilutions for 40 test compounds, as well as two 16-pointserial dilutions of staurosporine as reference compound, plus 16 high-and 16 low controls. Liquid handling and incubation steps were done on aautomated workstation equipped with a Innovadyne Nanodrop Express.

The assay plates were prepared by addition of 50 mL per well of compoundsolution in 90% DMSO. The kinase reactions were started by stepwiseaddition of 4.5 μl per well of a 2× peptide/ATP-solution and 4.5 μl perwell of a 2× enzyme solution. The final concentration of reagents duringkinase reaction were: 50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20,0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodiumorthovanadate. The peptide substrate used in the PKC-alpha and PKC-thetaassays was Dy495-X5-ME-Mpr-RFARKGSLRQKNV-COOH. Both enzymes were fulllength human recombinant protein expressed in insect cells (InvitrogenAG, Basel, Switzerland). Other components were adjusted specifically forthe respective kinase assays: PKC-alpha: 12 pM enzyme, 17 μM ATP, 1 μMpeptide substrate, 7 mM MgCl₂, 0.2 mM CaCl₂. PKC-theta: 29 pM enzyme, 70μM ATP, 1 μM peptide substrate, 7 mM MgCl₂, 0.2 mM CaCl₂.

Kinase reactions were incubated at 30° C. for 60 minutes andsubsequently terminated by addition of 16 μl per well of stop solution(100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA,and 0.015% Brij35).

Plates with terminated kinase reactions were transferred to the CaliperLC3000 workstations for reading. Phosphorylated and unphosphorylatedpeptides were separated using the Caliper microfluidic mobility shifttechnology and Kinase activities were calculated from the amounts offormed phospho-peptide.

Assay Example 1 Example 3 PKCα (IC₅₀ in nM) 1677 0.4 PKCθ (IC₅₀ in nM)462 0.2

Test results shown herein above and herein below may support the prodrugconcept of the compounds of the invention.

2. Bone Marrow Cell Proliferation (BM) Assay

Bone marrow cells from CBA mice (2.5×10⁴ cells per well in flat bottomtissue culture microtiter plates) were incubated in 100 μL RPMI mediumcontaining 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin (GibcoBRL, Basel Switzerland), 50 μM 2-mercaptoethanol (Fluka, Buchs,Switzerland), WEHI-3 conditioned medium (7.5% v/v) and L929 conditionedmedium (3% v/v) as a source of growth factors and serially dilutedcompounds. Seven three-fold dilution steps in duplicates per testcompounds were performed. After four days of incubation 1 μCi³H-thymidine was added. Cells were harvested after an additionalfive-hour incubation period, and incorporated ³H-thymidine wasdetermined according to standard procedures. Conditioned media wereprepared as follows. WEHI-3 cells (ATCC TIB68) and L929 cells (ATCCCCL 1) were grown in RPMI medium until confluence for 4 days and oneweek, respectively. Cells were harvested, resuspended in the sameculture flasks in medium C containing 1% FCS (Schreier and Tess 1981)for WEHI-3 cells and RPMI medium for L929 cells and incubated for 2 days(WEHI-3) or one week (L929). The supernatant was collected, filteredthrough 0.2 μm and stored in aliquots at −80° C. Cultures without testcompounds and without WEHI-3 and L929 supernatants were used as lowcontrol values. Low control values were subtracted from all values. Highcontrols without any sample were taken as 100% proliferation. Percentinhibition by the samples was calculated and the concentrations requiredfor 50% inhibition (IC₅₀ values) were determined.

Example 1 Example 3 IC₅₀ in nM 6741 ± 1117 1672 ± 256

B. In Vivo Administration of Compound of Example No. 1

A single dose of the compound of example 1 (3.0 mg/kg) was administeredp.o. to 3 male Beagle dogs. Compound I was dosed as an aqueoussuspension of the crystalline monohydrate form in Methylcellulose(0.5%): Tween 80 (1%) (90:10). Blood was taken in regular intervals byvenipuncture, and the samples were analyzed for a period of up to 24hours. The compounds of the examples 1, 2, and 3 were quantitativelyassessed over time, and the results are tabulated below:

Compound 1 Compound 2 Compound 3 Time (h) (nM) (nM) (nM) 0 — — — 0.083 —10.0 15.0 0.25 3.1 39.6 166.7 0.5 1.1 31.3 757.4 0.75 — 19.1 1198.0 1 —6.3 1308.3 2 — 7.7 1118.6 3 — 1.0 958.9 4 — — 762.0 7 — — 403.1 24 — —67.9

Key pharmacokinetic parameters (mean values±standard deviations (n=3)for 1, 2 and 3 after oral dosing of example No. 1.

Parameters Compound 1 Compound 2 Compound 3 C_(max) d.n. (nM) — 15 ± 8 441 ± 165 T_(max) (h) — 0.4 ± 0.1 0.9 ± 0.1 AUC d.n. (nM · h) — low 3350± 1402

Administration of Compound of Example No. 3

Compound of Example No. 3 was administered orally as the mono acetatesalt in a hard gelatine capsule to 6 fasted male Beagle dogs. A nominaldose of 100 mg/dog was given, resulting in dose of 8.9-11.3 mg/kg(weight of the dogs ranging from 8.9-11.3 kg). Blood was taken byvenipuncture, and sampling was performed up to 32 h. Bioanalyticdetermination was performed for 3 and is tabulated below:

Time (h) Compound 3 (nM) 0 — 0.25 177 0.5 936 1 2155 2 2548 3 2000 41898 6 1486 8 1238 24 276 32 116

Key pharmacokinetic parameters (mean values or range) for compound No. 3after oral dosing as described above.

Parameters Compound 3 C_(max) d.n. (nM)  323 T_(max) (h) 0.5-2 AUC d.n.(nM · h) 2790Physicochemical SectionSolubility Assessments

Solubility of compound Example No. 1 in simulated gastric and insimulated intestinal fluids at room temperature

Media Solubility [mg/mL] Final pH Simulated gastric fluid (SGF) 0.042.22 pH 2 Fasted state simulated 0.1 6.59 intestinal fluid (FaSSIF) pH6.5 Fed state simulated intestinal 0.13 6.05 fluid (FeSSIF) pH 5.8

Solubility of compound Example No. 3 (free form/acetate salt form) insimulated gastric fluids:

Free Form Acetate Salt Solubility Solubility Final Media [mg/ml] FinalpH [mg/ml] pH Simulated gastric fluid (SGF) 0.12 — 0.15 5.45 pH 2 Fastedstate simulated 0.03 — 0.05 3.97 intestinal fluid (FaSSIF) pH 6.5 Fedstate simulated intestinal 0.28 — 0.55 6.5 fluid (FeSSIF) pH 5.8Stability AssessmentsStability of Cpd. Example No. 1 in Gastric and Intestinal SimulatedFluids at 37° C.:

Amount in Area % of Media Time (hours) compound example No. 1 Simulatedgastric fluid (SGF) 0 95.9 pH 2 1 95.5 2.5 95.3 4.2 95.6 7 95.6 Fastedstate simulated 0 95.1 intestinal fluid (FaSSIF) pH 1 93.8 6.5 2.5 92.34.2 90.5 7 87.7 Fed state simulated intestinal 0 95.0 fluid (FeSSIF) pH5.8 1 94.9 2.5 94.8 4.2 94.8 7 94.8Stability of Compound Example No. 3 (Free Form/Acetate Salt Form) inGastric and Intestinal Simulated Fluids at 37° C.:

Free Form Acetate Salt Amount in Amount in Area % Area % of compound ofcompound Media Time (hours) example No. 3 example No. 3 Simulatedgastric 0 98.8 100 fluid (SGF) pH 2 1 98.6 96.4 2.5 98.1 93.2 4 97.992.4 7 97.9 90.0 Fasted state 0 100 100 simulated intestinal 1 100 98.6fluid (FaSSIF) pH 6.5 2.5 100 98.7 4 90.7 95.1 7 82.5 88.5 Fed statesimulated 0 100 100 intestinal fluid 1 100 99.1 (FeSSIF) pH 5.8 2.5 10098.9 4 99.2 98 7 98.7 97.5Utility Section

The compounds of the present invention are typically useful in theprevention or treatment of disorders or diseases where PKC, or mediatorsof other kinases play a role, for example in diseases or disordersmediated by T lymphocytes, B lymphocytes, mast cells, eosinophils orcardiomyocytes, and hence are typically indicated in acute or chronicrejection of organ or tissue allo- or xenografts, graft-versus-hostdisease, host-versus-graft disease, atherosclerosis, cerebralinfarction, vascular occlusion due to vascular injury such asangioplasty, restenosis, fibrosis (especially pulmonary, but also othertypes of fibrosis, such as renal fibrosis), angiogenesis, hypertension,heart failure, chronic obstructive pulmonary disease, CNS disease suchas Alzheimer disease or amyotrophic lateral sclerosis, cancer,infectious disease such as AIDS, septic shock or adult respiratorydistress syndrome, ischemia/reperfusion injury e.g. myocardialinfarction, stroke, gut ischemia, renal failure or hemorrhage shock, ortraumatic shock.

The compounds of the invention are also useful in the treatment and/orprevention of acute or chronic inflammatory diseases or disorders orautoimmune diseases e.g. sarcoidosis, fibroid lung, idiopathicinterstitial pneumonia, obstructive airways disease, includingconditions such as asthma, intrinsic asthma, extrinsic asthma, dustasthma, particularly chronic or inveterate asthma (for example lateasthma and airway hyperreponsiveness), bronchitis, including bronchialasthma, infantile asthma, rheumatoid arthritis, osteoarthritis, systemiclupus erythematosus, nephrotic syndrome lupus, Hashimoto's thyroiditis,multiple sclerosis, myasthenia gravis, type I diabetes mellitus andcomplications associated therewith, type II adult onset diabetesmellitus, uveitis, nephrotic syndrome, steroid dependent andsteroid-resistant nephrosis, palmoplantar pustulosis, allergicencephalomyelitis, glomerulonephritis, psoriasis, psoriatic arthritis,atopic eczema (atopic dermatitis), allergic contact dermatitis, irritantcontact dermatitis and further eczematous dermatitises, seborrheicdermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysisbullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneouseosinophilias, acne, alopecia greata, eosinophilic fasciitis,atherosclerosis, conjunctivitis, keratoconjunctivitis, keratitis, vernalconjunctivitis, uveitis associated with Behcet's disease, herpetickeratitis, conical cornea, Sjoegren's syndrome, dystorphia epithelialiscorneae, keratoleukoma, ocular pemphigus, Mooren's ulcer, scleritis,Graves' ophthalmopathy, severe intraocular inflammation, inflammation ofmucosa or blood vessels such as leukotriene B4-mediated diseases,gastric ulcers, vascular damage caused by ischemic diseases andthrombosis, cardiac hypertrophy, ischemic bowel disease, inflammatorybowel disease (e.g. Crohn's disease or ulcerative colitis), necrotizingenterocolitis, renal diseases including interstitial nephritis,Goodpasture's syndrome hemolytic uremic syndrome and diabeticnephropathy, nervous diseases selected from multiple myositis,Guillain-Barre syndrome, Meniere's disease and radiculopathy, collagendisease including scleroderma, Wegener's granuloma and Sjogren'syndrome, chronic autoimmune liver diseases including autoimmunehepatitis, primary biliary cirrhosis and sclerosing cholangitis),partial liver resection, acute liver necrosis (e.g. necrosis caused bytoxins, viral hepatitis, shock or anoxia), cirrhosis, fulminanthepatitis, pustular psoriasis, Behcet's disease, active chronichepatitis, Evans syndrome, pollinosis, idiopathic hypoparathyroidism,Addison disease, autoimmune atrophic gastritis, lupoid hepatitis,tubulointerstitial nephritis, membranous nephritis, or rheumatic fever.

The compounds of the invention may also be useful for treating tumors,e.g. breast cancer, genitourinary cancer, lung cancer, gastrointestinalcancer, epidermoid cancer, melanoma, ovarian cancer, pancreas cancer,neuroblastoma, head and/or neck cancer or bladder cancer, or in abroader sense renal, brain or gastric cancer; in particular (i) a breasttumor; an epidermoid tumor, such as an epidermoid head and/or neck tumoror a mouth tumor; a lung tumor, for example a small cell or non-smallcell lung tumor; a gastrointestinal tumor, for example, a colorectaltumor; or a genitourinary tumor, for example, a prostate tumor(especially a hormone-refractory prostate tumor); or (ii) aproliferative disease that is refractory to the treatment with otherchemotherapeutics; or (iii) a tumor that is refractory to treatment withother chemotherapeutics due to multidrug resistance.

The compounds may also useful for treating tumors of blood and lymphaticsystem (e.g. Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt'slymphoma, AIDS-related lymphomas, malignant immunoproliferativediseases, multiple myeloma and malignant plasma cell neoplasms, lymphoidleukemia, acute or chronic myeloid leukemia, acute or chroniclymphocytic leukemia, monocytic leukemia, other leukemias of specifiedcell type, leukemia of unspecified cell type, other and unspecifiedmalignant neoplasms of lymphoid, haematopoietic and related tissues, forexample diffuse large cell lymphoma, T-cell lymphoma or cutaneous T-celllymphoma). Myeloid cancer includes e.g. acute or chronic myeloidleukaemia.

Where a tumor, a tumor disease, a carcinoma or a cancer are mentioned,also metastasis in the original organ or tissue and/or in any otherlocation are implied alternatively or in addition, whatever the locationof the tumor and/or metastasis.

Preferably the compounds of the present invention are in particularuseful in the prevention and/or treatment of a disease or a disordermediated by T lymphocytes such as acute or chronic rejection of organ ortissue allo- or xenografts, graft-versus-host disease, host-versus-graftdisease, multiple sclerosis, psoriasis, or rheumatoid arthritis.

Poor bioavailability of drug products is very often a limiting factorfor pharmaceutically effective ingredients. Moreover bioavailabilitymight be species dependent. For example a well absorbed drug in mice,rat or dog or the like may not translate into a proper bioavailabilityin men. The present invention addresses this problem by providing aprodrug compound of formula (I) producing a favorable bioavailabilityfor its parent compounds, in particular in men. For example, as shown inthe experimental section, the compound of example 1 is converted intocompound of example 3 which can be detected in the blood as the maincomponent shortly after administration (for example after around anhour), and hence demonstrates the effective and favorable conversioninto the parent compound.

For the above uses the required dosage will of course vary depending onthe mode of administration, the particular condition to be treated andthe effect desired. In general, satisfactory results are indicated to beobtained systemically at daily dosages of from about 0.02 to 25 mg/kgper body weight. An indicated daily dosage in the larger mammal, e.g.humans, may be typically in the range from about 0.2 mg to about 2 g,conveniently administered, for example, in divided doses up to fourtimes a day or in retard form. Suitable unit dosage forms for oraladministration may typically comprise from ca. 0.1 to 500 mg activeingredient.

The compounds of the invention may be administered by any conventionalroute, in particular parenterally, for example in the form of injectablesolutions or suspensions, enterally, e.g. orally, for example in theform of tablets or capsules, topically, e.g. in the form of lotions,gels, ointments or creams, or in a nasal or a suppository form. Topicaladministration may for example be to the skin. A further form of topicaladministration may be to the eye. Pharmaceutical compositions comprisinga compound of the invention in association with at least onepharmaceutical acceptable carrier or diluent may be manufactured inconventional manner by mixing with a pharmaceutically acceptable carrieror diluent.

The compounds of the invention may be administered in free form or inpharmaceutically acceptable salt form or in hydrate form, e.g. asindicated above. Such salts or hydrates may be prepared in conventionalmanner and may typically exhibit the same order of activity as the freecompounds.

In accordance with the foregoing, the present invention also provides:

(1) A compound of the invention or a pharmaceutically acceptable salt orhydrate thereof, for use as a pharmaceutical;

(2) A compound of the invention or a pharmaceutically acceptable salt orhydrate thereof, for use as a PKC inhibitor, for example for use in anyof the particular indications hereinbefore set forth;

(3) A pharmaceutical composition, e.g. for use in any of the indicationsherein before set forth, comprising a compound of the invention or apharmaceutically acceptable salt or hydrate thereof, together with oneor more pharmaceutically acceptable diluents or carriers therefor;(4) A method for the treatment or prevention of a disease or conditionin which PKC activation plays a role or is implicated, e.g. for thetreatment of any of particular indication hereinbefore set forth in asubject in need thereof which comprises administering to the subject aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt or hydrate thereof;(5) The use of a compound of the invention or a pharmaceuticallyacceptable salt or hydrate thereof, for the manufacture of a medicamentfor the treatment or prevention of a disease or condition in which PKCactivation plays a role or is implicated; e.g. as indicated above.Combinations

The compounds of the invention may be administered as the sole activeingredient or in conjunction with, e.g. as an adjuvant to, other drugse.g. in immunosuppressive or immunomodulating regimens or otheranti-inflammatory agents, e.g. for the treatment or prevention of allo-or xenograft acute or chronic rejection or inflammatory or autoimmunedisorders, a chemotherapeutic agent or an anti-infective agent, e.g. ananti-viral agent such as e.g. an anti-retroviral agent or an antibiotic.

For example, the compounds of the invention may be used in combinationwith a calcineurin inhibitor, e.g. cyclosporin A, ISA247 or FK 506; amTOR inhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779,ABT578, TAFA-93, AP23573, AP23464, AP23841, biolimus-7 or biolimus-9; anascomycin having immuno-suppressive properties, e.g. ABT-281, ASM981,etc.; corticosteroids; cyclophosphamide; azathioprene; methotrexate;leflunomide; mizoribine; mycophenolic acid or salt; mycophenolatemofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogueor derivative thereof; a PKC inhibitor, e.g. as disclosed in WO 02/38561or WO 03/82859, e.g. the compound of Example 56 or 70; a SIP receptoragonist or modulator, e.g. FTY720 optionally phosphorylated or an analogthereof, e.g.2-amino-2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl-1,3-propanedioloptionally phosphorylated or1-{4-[1-(4-cyclohexyl-3-trifluoromethyl-benzylmimino)-ethyl]-2-ethyl-benzyl}-azetidine-3-carboxylicacid or its pharmaceutically acceptable salts; immunosuppressivemonoclonal antibodies, e.g., monoclonal antibodies to leukocytereceptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45,CD52, CD58, CD80, CD86 or their ligands; other immunomodulatorycompounds, e.g. a recombinant binding molecule having at least a portionof the extracellular domain of CTLA4 or a mutant thereof, e.g. an atleast extracellular portion of CTLA4 or a mutant thereof joined to anon-CTLA4 protein sequence, e.g. CTLA4Ig (for ex. designated ATCC 68629)or a mutant thereof, e.g. LEA29Y; adhesion molecule inhibitors, e.g.LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4antagonists, e.g. natalizumab (ANTEGREN®); or antichennokine antibodiesor antichemokine receptor antibodies, or low molecular weight chemokinereceptor antagonists, e.g. anti MCP-1 antibodies.

A compound of the invention may also be used in combination with otherantiproliferative agents. Such antiproliferative agents include, but arenot limited to:

(i) aromatase inhibitors, e.g. steroids, especially exemestane andformestane and, in particular, non-steroids, especiallyaminoglutethimide, vorozole, fadrozole, anastrozole and, veryespecially, letrozole;

(ii) antiestrogens, e.g. tamoxifen, fulvestrant, raloxifene andraloxifene hydrochloride;

(iii) topoisomerase I inhibitors, e.g. topotecan, irinotecan,9-nitrocamptothecin and the macromolecular camptothecin conjugatePNU-166148 (compound A1 in WO99/17804);

(iv) topoisomerase II inhibitors, e.g. the anthracyclines doxorubicin(including liposomal formulation, e.g. CAELYXT™), epirubicin, idarubicinand nemorubicin, the anthraquinones mitoxantrone and losoxantrone, andthe podophillotoxines etoposide and teniposide;(v) microtubule active agents, e.g. the taxanes paclitaxel anddocetaxel, the vinca alkaloids, e.g., vinblastine, especiallyvinblastine sulfate, vincristine especially vincristine sulfate, andvinorelbine, discodermolide and epothilones, such as epothilone B and D;(vi) alkylating agents, e.g. cyclophosphamide, ifosfamide and melphalan;(vii) histone deacetylase inhibitors;(viii) farnesyl transferase inhibitors;(ix) COX-2 inhibitors, e.g. celecoxib (Celebrex®), rofecoxib (Vioxx®)and lumiracoxib (COX189);(x) MMP inhibitors;(xi) mTOR inhibitors;(xii) antineoplastic antimetabolites, e.g. 5-fluorouracil, tegafur,capecitabine, cladribine, cytarabine, fludarabine phosphate,fluorouridine, gemcitabine, 6-mercaptopurine, hydroxyurea, methotrexate,edatrexate and salts of such compounds, and furthermore ZD 1694(RALTITREXED™), LY231514 (ALIMTA™), LY264618 (LOMOTREXOL™) and OGT719;(xiii) platin compounds, e.g. carboplatin, cis-platin and oxaliplatin;(xiv) compounds decreasing the protein kinase activity and furtheranti-angiogenic compounds, e.g. (i) compounds which decrease theactivity of the Vascular Endothelial Growth Factor (VEGF) (b) theEpidermal Growth Factor (EGF), c-Src, protein kinase C, Platelet-derivedGrowth Factor (PDGF), Bcr-Abl tyrosine kinase, c-kit, Flt-3 andInsulin-like Growth Factor I Receptor (IGF-IR) and Cyclin-dependentkinases (CDKs); (ii) Imatinib, midostaurin, Iressa™ (ZD1839), CGP 75166,vatalanib, ZD6474, GW2016, CHIR-200131, CEP-7055/CEP-5214, CP-547632 andKRN-633; (iii) thalidomide (THALOMID), celecoxib (Celebrex), SU5416 andZD6126;(xv) gonadorelin agonists, e.g. abarelix, goserelin and goserelinacetate;(xvi) anti-androgens, e.g. bicalutamide (CASODEX™);(xvii) bengamides;(xviii) bisphosphonates, e.g. etridonic acid, clodronic acid, tiludronicacid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acidand zoledronic acid;(xix) antiproliferative antibodies, e.g. trastuzumab (Herceptin™),Trastuzumab-DM1, erlotinib (Tarceva™), bevacizumab (Avastin™), rituximab(Rituxan®), PRO64553 (anti-CD40) and 2C4 Antibody;(xx) temozolomide (TEMODAL®);(xxi) Statins.

The structure of the active agents identified by code nos., generic ortrade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications).

In accordance with the foregoing the present invention further provides:

(6) A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount ofa) a compound of formula I or a pharmaceutically acceptable salt orhydrate thereof, and b) a second drug substance, said second drugsubstance being for example for use in any of the particular indicationshereinbefore set forth;(7) A combination, e.g. a kit, comprising a therapeutically effectiveamount of a compound of formula I or a pharmaceutically acceptable saltor hydrate thereof, and a second drug substance, said second drugsubstance being for example as disclosed above. Where a compound of theinvention is administered in conjunction with otherimmunosuppressive/immunomodulatory, anti-inflammatory or antineoplasticagent, e.g. as disclosed above, dosages of the co-administered drug oragent will of course vary depending on the type of co-drug or -agentemployed, or the specific drug or agent used, or the condition beingtreated and so forth.

In another embodiment there is provided a method of manufacturing acompound of formula (I),

wherein X is CH or N; R is H or PO₃H₂; R1 is H or C₁₋₄alkyl; R2 is H orC₁₋₄alkyl; R3 is H, C₁₋₄alkyl, CN, Hal or OH; and R4 and R5 areindependently from each other H, or C₁₋₄alkyl; or R4 and R5 formtogether with the carbon atom to which they are attached a 3-6 memberedcycloalkyl group, comprising:(a) optionally, for compounds wherein R1 and/or R2 are hydrogen,treating the maleimide of formula (Va) for example with di-tert-butyldicarbonate in the presence or absence of a solvent such as THF ordichloromethane and/or a base such as triethyl amine, thereby yieldingthe maleimide of formula (Va) comprising tert-butoxycarbonyl groupsinstead of the hydrogens on R1 and/or R2 where appropriate;(b) treating the optionally protected maleimide of formula (Va) forexample with formaldehyde in the presence or absence of a solvent and/ora base such as potassium carbonate, thereby yielding an alcohol offormula (Vb), wherein R=H;(c) optionally treating the alcohol of formula (Vb) for example withtrichloroacetonitrile typically in the presence of a base, e.g. DBU ortrimethylamine to form a reactive ester, followed by the treatment witha phosphorylating agent, e.g. with a phosphoric acid ester, e.g. withphosphoric acid di-tert-butyl ester typically in the presence of a base,e.g. DBU or trimethylamine, whereupon the resulting intermediate esteris treated with an appropriate acid, e.g. hydrochloric acid or TFA inthe absence of presence of a solvent such as THF, dichloromethane,dichloroethane or the like to yield the final product in accordance togeneral formula (I), or as an alternative step (c)alcohol of formula (Vb) may be reacted directly with a phosphoric acidester, e.g. with phosphoric acid di-tert-butylester, e.g. underMitsunobu reaction conditions to furnish the phosphoric acid ester,which may then be hydrolyzed, e.g. with trifluoroacetic acid, e.g. indichloromethane to furnish the final product of formula (I).

The invention claimed is:
 1. A compound of formula (I) or apharmaceutically acceptable salt or hydrate thereof,

Wherein X is CH or N; R is H or PO₃H₂; R1 is H; or C₁₋₄alkyl; R2 is H;or C₁₋₄alkyl; R3 is H; C₁₋₄alkyl; CN; Hal; or OH; and R4 and R5 areindependently from each other H, or C₁₋₄alkyl; or R4 and R5 formtogether with the carbon atom to which they are attached a 3-6 memberedcycloalkyl group.
 2. The compound of formula (I) according to claim 1 ora pharmaceutically acceptable salt or hydrate thereof, wherein X is CH;R is H; R1 is H; R2 is H; or C₁₋₄alkyl; R3 is H; or C₁₋₄alkyl; and R4and R5 are independently from each other H; or R4 and R5 form togetherwith the carbon atom to which they are attached a 3-6 memberedcycloalkyl group.
 3. The compound of formula (I) according to claim 1 ora pharmaceutically acceptable salt or hydrate thereof, wherein X is N; Ris PO₃H₂; R1 is H; R2 is H; or C₁₋₄alkyl; R3 is H; and R4 and R5 areindependently from each other H; or R4 and R5 form together with thecarbon atom to which they are attached a 3-6 membered cycloalkyl group.4. The compound according to claim 1 which is a compound of formula(III)

or a pharmaceutically acceptable salt or hydrate thereof.
 5. Thecompound in accordance to claim 1, which is phosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester,mono-hydrate.
 6. The compound in accordance to claim 1, which is3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-1-hydroxymethyl-4-(7-methyl-1H-indol-3-yl)-pyrrole-2,5-dioneor a pharmaceutically acceptable salt thereof.
 7. The compound inaccordance to claim 1, which is phosphoric acidmono-{3-(1H-indol-3-yl)-4-[2-(4-methyl-piperazin-1-yl)-quinazolin-4-yl]-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl}ester or a pharmaceutically acceptable salt thereof.
 8. A crystallineform of phosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester,which has an X-ray powder diffraction pattern with at least one of thefollowing peaks at an angle of refraction 2 theta (θ) of 9.525, 16.356,17.091, 18.005, 20.859, each ±0.2, especially as depicted in FIG. 1and/or Table
 1. 9. The crystalline form of phosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester,according to claim 8, in the form of the mono-hydrate, which has anX-ray powder diffraction pattern with at least one of the followingpeaks at an angle of refraction 2 theta (θ) of 9.525, 16.356, 17.091,18.005, 20.859, each ±0.2, especially as depicted in FIG. 1 and/orTable
 1. 10. The crystalline form of phosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester,according to claim 8, which has an X-ray powder diffraction pattern withat least three of the following peaks at an angle of refraction 2 theta(θ) of 9.525, 16.356, 17.091, 18.005, 20.859, each ±0.2, especially asdepicted in FIG. 1 and/or Table
 1. 11. The crystalline form ofphosphoric acidmono-[3-[3-(4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-(7-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]ester,according to claim 8, which has an X-ray powder diffraction pattern withall the following peaks at an angle of refraction 2 theta (θ) of 9.525,16.356, 17.091, 18.005, 20.859, each ±0.2, especially as depicted inFIG. 1 and/or Table
 1. 12. A combination, comprising a therapeuticallyeffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt or hydrate thereof, and a second drug substance selectedfrom selected from calcineurin inhibitor a mTOR inhibitor; an ascomycinhaving immuno-suppressive properties; cyclophosphamide; azathioprene;methotrexate; leflunomide; mizoribine; mycophenolic acid or salt;mycophenolate mofetil; 15-deoxyspergualine or an immunosuppressivehomologue, analogue or derivative thereof; a PKC inhibitor, a S1Preceptor agonist or modulator, immunosuppressive monoclonal antibodies,other immunomodulatory compounds; adhesion molecule inhibitors, LFA-1antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4antagonists; or antichemokine antibodies or antichemokine receptorantibodies, or low molecular weight chemokine receptor antagonists; oranother antiproliferative agent selected from (i) aromatase inhibitors,(ii) antiestrogens, (iii) topoisomerase I inhibitors, (iv) topoisomeraseII inhibitors; (v) microtubule active agents; (vi) alkylating agents;(vii) histone deacetylase inhibitors; (viii) farnesyl transferaseinhibitors; (ix) COX-2 inhibitors; (x) MMP inhibitors; (xi) mTORinhibitors; (xii) antineoplastic antimetabolites; (xiii) platincompounds; (xiv) compounds decreasing the protein kinase activity andfurther anti-angiogenic compounds; (xv) gonadorelin agonists; (xvi)anti-androgens; (xvii) bengamides; (xviii) bisphosphonates; (xix)antiproliferative antibodies; (xx) temozolomide (TEMODAL®); (xxi)Statins.
 13. A method of manufacturing a compound of formula (I) asdefined in claim 1,

wherein X is CH or N; R is H or PO₃H₂; R1 is H; or C₁₋₄alkyl; R2 is H;or C₁₋₄alkyl; R3 is H; C₁₋₄alkyl; CN; Hal; or OH; and R4 and R5 areindependently from each other H, or C₁₋₄alkyl; or R4 and R5 formtogether with the carbon atom to which they are attached a 3-6 memberedcycloalkyl group, comprising: (a) optionally, for compounds wherein R1and/or R2 are hydrogen, treating the maleimide of formula (Va) withdi-tert-butyl dicarbonate in the presence or absence of a solvent suchas THF or dichloromethane and/or a base such as triethyl amine, therebyyielding the maleimide of formula (Va) comprising tert-butoxycarbonylgroups instead of the hydrogens on R1 and/or R2 where appropriate; (b)treating the optionally protected maleimide of formula (Va) withformaldehyde in the presence or absence of a solvent and/or a base,thereby yielding an alcohol of formula (Vb), wherein R=H; (c) optionallytreating the alcohol of formula (Vb) with trichloroacetonitriletypically in the presence of a base, to form a reactive ester, followedby the treatment with a phosphorylating agent, typically in the presenceof a base, whereupon the resulting intermediate ester is treated with anappropriate acid, in the absence of presence of a solvent to yield thefinal product in accordance to general formula (I), or as an alternativestep (c) alcohol of formula (Vb) may be reacted directly with aphosphoric acid ester, under Mitsunobu reaction conditions to furnishthe phosphoric acid ester, which may then be hydrolyzed, to furnish thefinal product of formula (I)